1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the inhibition and/or inactivation of nucleases which can degrade DNA (deoxyribonucleases) and/or RNA (ribonucleases). Inhibition and/or inactivation of nucleases in the present invention employs at least one, and in many cases at least two nuclease inhibitors. These nuclease inhibitors include anti-nuclease antibodies and non-antibody nuclease inhibitors.
2. Description of Related Art
The quality of an RNA preparation greatly affects the results obtained when analyzing it by a number of different molecular biology techniques such as northern blotting, ribonuclease protection assays and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). Degraded RNA will produce a lower signal than in an equivalent intact RNA sample.
RNA is much more susceptible to degradation than DNA (Sambrook et al., 1989). RNA is readily hydrolyzed when exposed to conditions of high pH, metal cations, high temperatures and contaminating ribonucleases. A major cause of RNA degradation is ribonuclease contamination, and this must be guarded against in virtually all RNA-related procedures, including RNA isolation, mRNA purification, RNA storage, northern blotting, nuclease protection assays, RT-PCR, in vitro transcription and/or translation and RNA diagnostics. In addition to the endogenous ribonucleases from cells and tissues, finger grease and bacteria and/or fungi in airborne dust particles are common sources of ribonuclease. To minimize ribonuclease contamination, appropriate precautions must be followed when handling RNA (Blumberg, 1987; Wu, 1997).
Ribonucleases are difficult to inactivate. For example, bovine pancreatic ribonuclease A (RNase A) has no activity at 90xc2x0 C. However, if the enzyme is quickly cooled to 25xc2x0 C., the activity is fully restored. This process is known as reversible thermal denaturation. If the RNase A is incubated at 90xc2x0 C. over time, then decreasing fractions of the activity are recovered at 25xc2x0 C. This process is known as irreversible thermoinactivation. At 90xc2x0 C., it takes several hours to inactivate RNase A (Zale and Klibanov, 1986). Much of the lost activity is attributed to disulfide interchange (Zale and Klibanov, 1986). Further, the inventors and others have found that ribonucleases can even withstand autoclaving (121xc2x0 C., 15 psi, 15 minutes) to some degree. Spackman et al. (1960) tested the stability of RNase A and concluded that it was stable to heat, extremes of pH, and the protein denaturant, urea, results emphasizing the difficulty researchers have had inactivating ribonucleases. For the above reason, a variety of methods other than heating have been developed to inhibit or inactivate ribonucleases. These methods, and their disadvantages, are described below.
In one method of destroying RNases, diethyl pyrocarbonate (DEPC) is added to final concentration of 0.1% to molecular biology reagents, glassware or electrophoresis apparatus, followed by incubating at 37xc2x0 C. for several hours and then autoclaving for 15-20 minutes to destroy the DEPC (Wolf et al., 1970). DEPC reacts with the xcex5-amino groups of lysine and the carboxylic groups of aspartate and glutamate both intra- and intermolecularly (Wolf et al., 1970). This chemical reaction forms polymers of the ribonuclease. However, there are several disadvantages to using DEPC: (1) It is a possible carcinogen and is hazardous to humans; (2) some commonly used molecular biology reagents such as Tris react with and inactivate DEPC; (3) treatment of samples with DEPC is time-consuming; (4) DEPC reacts with the adenine residues of RNA, rendering it inactive in in vitro translation reactions (Blumberg, 1987) and 5) If all of the DEPC is not destroyed by autoclaving, remaining trace amounts may inhibit subsequent enzymatic reactions.
Traditionally, RNA is stored in DEPC-treated water or TE buffer. However, the RNA is not protected from degradation if the sample or the storage solution has a minor ribonuclease contamination. It has been suggested that RNA be stored in ethanol, formamide, or guanidinium to protect an RNA sample from degradation because these environments minimize ribonuclease activity (Chomczynski, 1992; Gilleland and Hockett, 1992). The obvious disadvantage is that the RNA sample cannot be directly utilized for analysis or enzymatic reactions unless the ethanol, formamide, or guanidinium is removed.
Guanidinium thiocyanate is commonly used to inhibit RNases during RNA isolation (Chomczynski and Sacchi, 1987; Sambrook et al., 1989). A high concentration of guanidinium thiocyanate combined with P-mercaptoethanol is used to isolate RNA from tissues, even those that are rich in ribonucleases, such as pancreas (Chirgwin et al., 1979). Guanidinium is an effective inhibitor of most enzymes due to its chaotropic nature. However, if RNA is dissolved in guanidinium, then it must first be purified from the guanidinium prior to being used in an enzymatic reaction.
Vanadyl-ribonucleoside complexes (VRC) may be used to inhibit RNases during RNA preparation (Berger and Birkenmeier, 1979). The drawback to using VRC, is that VRC strongly inhibits the translation of mRNA in cell-free systems and must be removed from RNA samples by phenol extraction (Sambrook et al., 1989).
Favaloro et al. (1980) employed macaloid, a clay, to absorb RNases. A limitation of this method is that it is difficult to completely remove the clay from RNA samples. Other reagents have been used to inhibit ribonucleases including sodium dodecylsulfate (SDS), ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite and ammonium sulfate (Allewell and Sama, 1974; Jocoli and Ronald, 1973; Lin, 1972; Jones, 1976; Mendelsohn and Young, 1978). None of these reagents are strong inhibitors alone. Like many of the RNase inhibitors already described, although these chemicals inhibit RNase activity, they also may inhibit other enzymes such as reverse transcriptase and DNase I. Therefore, the RNA must be purified away from the inhibitory reagent(s) before it can be subjected to other enzymatic processes.
Two types of proteinaceous RNase inhibitors are commercially available: human placental ribonuclease inhibitor (Blackburn et al., 1977) and PRIME Inhibitor(trademark) (Murphy et al., 1995). RNases of the class A family bind tightly to these protein inhibitors and form noncovalent complexes that are enzymatically inactive. The major disadvantage of these inhibitors is that they have a narrow spectrum of specificity. They do not inhibit other classes of RNases. Another disadvantage when using placental ribonuclease inhibitor is that it denatures within hours at 37xc2x0 C., releasing the bound ribonuclease.
Heat has been used to inactivate RNase A by mediating the breakage of disulfide bonds. Zale and Klibanov (1986) performed inactivation of RNase A at 90xc2x0 C. and pH 6.0 for 1 hour, which induced the following chemical changes: disulfide interchange, xcex2-elimination of cysteine residues, and deamidation of asparagine. This type of heat treatment did not completely inactivate the ribonuclease. A major disadvantage is that a long-term, high-temperature treatment (90-100xc2x0 C.) is incompatible with RNA. Such treatment promotes the hydrolysis of RNA. In fact, the inventors have found that total RNA incubated at 65xc2x0 C. for several hours is almost completely degraded. Thus, treating an RNase sample with extreme heat to inactivate ribonucleases will mediate the destruction of the RNA which the user is trying to protect.
The present invention provides methods and compositions for rapidly inhibiting and/or inactivating nucleases. In some aspects, the present invention relates to methods for inhibiting nucleases with one or more nuclease inhibitors and compositions comprising nuclease inhibitors that can be used in such methods.
Such methods may comprise: a) obtaining at least a first nuclease inhibitor; b) obtaining at least a second nuclease inhibitor; c) obtaining a composition; and d) admixing the nuclease inhibitors and the composition. Under circumstances where there are nucleases present in the composition, those nucleases are inhibited to some extent. Note that complete inactivation or inhibition of nucleases is not required to obtain benefit from the invention. Further, note that these aspects of the invention are not limited to cases where one knows that there is a nuclease present in the composition. Therefore, it is entirely within the scope of the invention to use the inhibitors disclosed herein to treat a composition which may have nucleases in it, or even a composition which a research is confident does not have nucleases in it, but which the researcher wishes to treat out of an abundance of caution. In some embodiments, the composition will have a nuclease present, for example a ribonuclease (RNase), deoxyribonuclease (DNase), a non-specific nuclease, or a combination of two or more of these, as described elsewhere in this specification. Some embodiments involve inhibition of RNase, DNase, a non-specific nuclease, or a combination thereof.
In some broad aspects, the present invention relates to methods and compositions for inhibiting or inactivating nucleases using at least two nuclease inhibitors, which can include, in various embodiment, anti-nuclease antibodies and non-antibody nuclease inhibitors. A composition comprising at least two nuclease inhibitors is referred to herein as a xe2x80x9cnuclease inhibitor cocktail,xe2x80x9d and includes but is not limited to, the specific inhibitors disclosed elsewhere in this specification. The composition treated according to the methods of the invention can be any composition that one of skill in the art would find it beneficial to treat in order to prevent nuclease activity in the composition. In most cases, the composition will be liquid, although solid compositions such as a matrix comprising immobilized nuclease inhibitor may be treated as well. In some preferred embodiments, the composition is a reagent used in molecular biology.
The methods of the invention frequently involve the preparation of a nuclease inhibitor cocktail by the mixing of the first and second nuclease inhibitors. Such a cocktail may be mixed with the composition at any time. For example, the cocktail may be prepared and then relatively immediately mixed with the composition. Alternatively, the cocktail may be prepared in advance. In many cases, the cocktail may be prepared and provided as a commercial product to a researcher, and the researcher practices the invention by obtaining the first and second nuclease inhibitors in the form of the cocktail and then admixing the cocktail with the composition.
In many embodiments of the application, the composition comprises a nucleic acid, for example, DNA or RNA. In some embodiments, the composition comprises RNA that has been purified. For example he composition is further defined as an in vitro translation reaction or a transcription reaction. In some, more specific embodiments, the composition comprises both DNA and RNA. In additional embodiments, the composition is or comprises a reagent used in molecular biology. Exemplary such reagents can be water, tris-EDTA buffer (TE), sodium chloride/sodium citrate buffer (SSC), MOPS/sodium acetate/EDTA buffer (MOPS), Tris buffer, ethylenediamine tetraacetic acid (EDTA), nucleic acid hybridization buffer, sodium acetate buffer, DNase 1 digestion buffer, transcription buffer, reverse transcription buffer, cell free extract for in vitro translation, in situ hybridization buffer, or nucleic acid storage buffer/solution.
In some preferred embodiments, the first nuclease inhibitor or the second nuclease inhibitor is an anti-nuclease antibody. Such antibodies can be a polyclonal or monoclonal antibodies, with some presently preferred commercial embodiments of the invention employing polyclonal antibodies. The anti-nuclease antibodies may be anti-ribonuclease antibodies, anti-deoxyribonuclease antibodies, or antibodies to non-specific nucleases. The invention may comprise the use of any number of anti-nuclease antibodies with various specificities. The invention includes the use of a mixture of two anti-nuclease antibodies that each inhibit a different nuclease. However, mixtures of three, four, or more anti-nuclease antibodies, each of which inhibit different or the same nucleases, may be used.
Many embodiments involve one or more anti-ribonuclease antibodies. Such anti-ribonuclease antibodies may be an antibody capable of binding to one or more of RNase A, a member of the RNase A family, RNase B, RNase C, RNase 1, RNase T1, RNase T2, RNase L, a member of the RNase H family, a member of the angiogenin RNase family, eosinophil RNase, a micrococcal nuclease, a member of the mammalian ribonuclease 1 family, a member of the ribonuclease 2 family, a messenger RNA ribononuclease, 5xe2x80x2-3xe2x80x2 exoribonuclease, 3xe2x80x2-5xe2x80x2 exoribonuclease, a decapping enzyme, a deadenylase, RNase P, RNase III, RNase E, RNase I,I*, RNase HI, RNase HII, RNase M, RNase R, RNase IV, F; RNase P2,0, PIV, PC, RNase N, RNase II, PNPase, RNase D, RNase BN, RNase T, RNase PH, OligoRNase, RNase R, RNase H, RNase Sa, RNase F1, RNase U2, RNase Ms, RNase St, or RNase P. Some presently preferred anti-ribonuclease antibodies are those that inhibit one or more members of the RNase A family, for example an anti-RNase A antibody. Others are anti-RNase 1 antibodies and anti-RNase T1 antibodies.
Some embodiments involve one or more anti-deoxyribonuclease antibodies, for example, anti-DNase 1 antibodies. Some involve antibodies capable of binding to a non-specific nuclease such as S1 nuclease or micrococcal nuclease. The non-specific nucleases may degrade both RNA and DNA.
In some embodiments, both the first nuclease inhibitor and the second nuclease are anti-nuclease antibodies. Of course, third, fourth, fifth, sixth, and/or more nuclease inhibitors may be used in combination, and all of these may be anti-nuclease antibodies. Some embodiments involve a combination of nuclease inhibitors comprising both antibody inhibitors and non-antibody inhibitors. Many methods will comprise the use of an anti-RNase A antibody. In a preferred embodiment, the anti-RNase A antibody will be an antibody that can bind and inhibit any RNase of the RNase A superfamily, for example, RNase A, RNase B, and RNase C. Some presently preferred embodiments, involve a cocktail comprising at least an anti-RNase A antibody, an anti-RNase 1 antibody, and an anti-RNase T1 antibody.
The first nuclease inhibitor, the second nuclease inhibitor, and/or any other nuclease inhibitors involved in the compositions and methods of the invention may be non-antibody based inhibitors, such as those described elsewhere in the specification. For example, such inhibitors may be is human placental ribonuclease inhibitor, a bovine ribonuclease inhibitor, a porcine ribonuclease inhibitor, diethyl pyrocarbonate, ethanol, formamide, guanidinium thiocyanate, vanadyl-ribonucleoside complexes, macaloid, sodium dodecyl sulfate (SDS), ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), xcex2-mercaptoethanol, cysteine, dithioerythritol, tris (2-carboxyethyl) phosphene hydrochloride, or a divalent cation such as Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, or Cu+2. Certain embodiments involve the use of human placental ribonuclease inhibitor.
Other embodiments involve solutions comprising at least a first nuclease inhibitor and a second nuclease inhibitor. Such solutions may be xe2x80x9cnuclease inhibitor cocktails,xe2x80x9d xe2x80x9cribonuclease (or RNase) inhibitor cocktails,xe2x80x9d xe2x80x9cdeoxyribonuclease (or DNase) inhibitor cocktails,xe2x80x9d etc., as appropriate based on their activities. Such solutions may comprise a nucleic acid molecule. In some embodiments, the solution is a reagent used in molecular biology, such as described above.
The first and second nuclease inhibitors in such solutions may be any of the anti-nuclease antibodies or non-antibody based inhibitors described above and elsewhere in this specification. In some embodiments, the solution will comprise at least a third anti-nuclease antibody, although any number of such antibodies may be employed. Some presently commercially preferred embodiments comprise at least an anti-RNase A antibody, an anti-RNase 1 antibody, and an anti-RNase T1 antibody, and may further comprise an anti-RNase II antibody, an anti-eosinophil antibody, and an anti-angiogenin antibody. In other preferred embodiments, the solution will comprise at least an anti-DNase antibody, an anti-S1 nuclease antibody, and an anti-micrococcal nuclease antibody. These and other particular embodiments may also comprise human placental ribonuclease inhibitor.
In one presently preferred commercial embodiment, the nuclease inhibitor cocktail is a mixture of anti-ribonuclease antibodies that bind and inactivate RNase A, B and C in addition to RNase 1 and RNase T1.
In some specific embodiments, the invention concerns methods of performing in vitro translation comprising obtaining a first nuclease inhibitor, which inhibitor is further defined as an anti-nuclease antibody, and placing the anti-nuclease antibody in an in vitro translation reaction. In many cases, the in vitro translation reaction comprises at least one nuclease, which may be a ribonuclease, a deoxyribonuclease, or a nonspecific nuclease, as described elsewhere in the specification. Of course, the reaction may further comprise a combination of two or more nucleases. The in vitro translation reaction will also comprise a nucleic acid, which will usually be RNA, in particular, the mRNA to be employed in translation. However, the reaction may also comprise DNA, for example, as the result of a cell-based isolation procedure or a coupled, linked, or separated transcription/translation reaction performed according to any of a number of methods known to those of skill in the art. The anti-nuclease antibody can be any of those described above. In many cases the methods involve obtaining a second nuclease inhibitor and placing the second nuclease inhibitor in the in vitro translation reaction. The second nuclease inhibitor can be any anti-nuclease antibody or non-antibody nuclease inhibitor as described elsewhere in the specification. In some preferred embodiments, the first nuclease inhibitor is an anti-micrococcal nuclease antibody and the second anti-nuclease antibody is an anti-ribonuclease antibody. These methods will often involve the use of cell-free translation systems, such as, for example, the reticulocyte lysate, wheat germ lysate, Drosophila lysate, yeast lysate, etc. systems known to those of skill in the art and described in the literature and elsewhere in the specification.
The invention also relates to kits for the performance of various microbiological procedures, which kits comprise the nuclease inhibitors described herein. These kits may contain either a single nuclease inhibitor, or multiple nuclease inhibitors. In some cases, these kits may contain a cocktail of nuclease inhibitors, as described elsewhere in the specification. In one particular embodiment, the kit is further defined as a kit for in vitro translation and comprises at least one anti-nuclease antibody and some or all of the necessary components for, or to make, a cell-free translation system. Such systems are known to those of skill in the art.
The methods and compositions of the present invention provide for rapidly inhibiting and/or inactivating nucleases using anti-nuclease antibodies, non-antibody nuclease inhibitors, or both. By employing the methods and compositions of the present invention, a sample of DNA or RNA maintains its intact, full-length form during production and storage.
The Present Invention
The present invention comprises methods and compositions for rapidly inhibiting and/or inactivating nucleases (deoxyribonucleases (DNases) and ribonucleases (RNases)) using at least two nuclease inhibitors. These nuclease inhibitors may be one or more anti-nuclease antibodies, one or more non-antibody nuclease inhibitors, or a combination of at least one anti-nuclease antibody and at least one non-antibody nuclease inhibitor. The non-antibody nuclease inhibitors may be proteinaceous inhibitors, such as Human Placental RNase Inhibitor (RIP), or non-proteinaceous inhibitors, such as divalent cations. Of course, one can determine other nuclease inhibitors by employing the methods disclosed.
In one presently preferred commercial embodiment, the nuclease inhibitor cocktail is a mixture of protein-based ribonuclease inhibitors that non-covalently bind and inactivate RNase A, B and C in addition to RNase 1 and RNase T1. This nuclease inhibitor cocktail is distinct from human placental ribonuclease inhibitor in that it has more robust interaction with RNases and does not release active RNases in the absence of dithiothreitol (DTT) or other reducing agents. It is an antibody-based, RNase-free mixture of different RNase inhibitors which can be useful in solving many RNase contamination problems. The nuclease inhibitor cocktail designed to block RNase A, B, C, RNase T1 and RNase 1 activities may be provided in some embodiments with final concentrations in the cocktail of 25 U/xcexcl (xcx9c9 mg/ml) of anti-RNase A, 4.5 mg/ml of anti-RNase 1 and 4.5 mg/ml of anti-RNase T1. However; any concentrations that accomplish the goals of the invention are within the scope of the invention.
The present invention can be employed to inhibit and/or inactivate nucleases, thereby providing reagents that are free of RNase activity and DNase activity. For example, the anti-RNase antibodies of the present invention have a broader spectrum than human placental RNase Inhibitor (RIP), inhibit common eukaryotic and prokaryotic nucleases (RNase A, B, C, RNase 1, T1, etc.), do not interfere with action of SP6, T7, and T3 polymerase, M-MLV Reverse Transcriptase or Taq DNA polymerase, are effective from pH 5.0 to 8.5, and are active from 37xc2x0 C. to 65xc2x0 C..
Definitions
As used herein, the term xe2x80x9cinhibitionxe2x80x9d of nuclease activity means that activity of at least one nuclease is reduced in a sample treated according to the invention relative to a sample not treated according to the invention. xe2x80x9cInhibitionxe2x80x9d does not require nuclease inactivation or even substantial nuclease inactivation. The term xe2x80x9csubstantial inhibitionxe2x80x9d connotes that there is no substantial degradation of DNA or RNA detected in a composition that may contain DNA or RNA. xe2x80x9cSubstantialxe2x80x9d degradation is defined as degradation that would impair the use of the DNA or RNA in the types of protocols described in this specification. As used herein, the terms xe2x80x9cnuclease inactivationxe2x80x9d or the xe2x80x9cinactivation of nucleasesxe2x80x9d denotes that there is no detectable degradation of the sample DNA or RNA under the assay conditions used, and that the nuclease is irreversibly rendered inoperative. The term xe2x80x9csubstantially inactivatedxe2x80x9d connotes that there is no substantial degradation of DNA or RNA detected in a composition that may contain DNA or RNA, and that the nuclease is irreversibly rendered inoperative.
A combination of at least two nuclease inhibitors is referred to herein as a xe2x80x9cnuclease inhibitor cocktail.xe2x80x9d The nuclease inhibitors include anti-nuclease antibodies and non-antibody nuclease inhibitors (xe2x80x9cother nuclease inhibitorsxe2x80x9d). xe2x80x9cIn vitro translationxe2x80x9d is a process of protein synthesis outside the living cell using cell-free extract and mRNA transcript as genetic material for translation. Examples of in vitro translation reactions include IVT Retic Lysate(trademark) or IVT Wheat Germ(trademark) (Ambion). Of course, those of ordinary skill in the art will understand how to perform in vitro translation reactions with other than the examples given.
xe2x80x9cIn vitro coupled transcription/translationxe2x80x9d is a process of protein synthesis in a cell-free lysate where the starting genetic material is DNA and both transcription and translation processes are proceeding simultaneously. An example of in vitro coupled transcription/translation is the PROTEINscript-PRO(trademark) system (Ambion). One of ordinary skill in the art will be able to perform in vitro coupled transcription/translation reactions with other than the example given.
An xe2x80x9cin vitro transcription reactionxe2x80x9d is the production of RNA from a DNA template under control of specific RNA polymerase(s) in the presence of rNTPs in the transcription buffer optimized for in vitro transcription. Examples of in vitro transcription reaction are MAXIscript(trademark), MEGAscript(trademark), and mMESSAGE mMACHINE(trademark) (Ambion). One of ordinary skill in the art will understand how to perform in vitro transcription reactions with other than the examples given.
In standard in vitro translation reactions, purified RNA is used as a template for translation. xe2x80x9cLinkedxe2x80x9d and xe2x80x9ccoupledxe2x80x9d systems, on the other hand, use DNA as a template. RNA is transcribed from the DNA and subsequently translated without any purification. Such systems typically combine a prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts to synthesize proteins from exogenous DNA templates. DNA templates for transcription:translation reactions may be cloned into plasmid vectors or generated by PCR. The xe2x80x9clinkedxe2x80x9d system is a two-step reaction, based on transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate. Because the transcription and translation reactions are separate, each can be optimized to ensure that both are functioning at their full potential.
Unlike eukaryotic systems where transcription and translation occur sequentially, in E. coli, transcription and translation occur simultaneously within the cell. In vitro E. coli translation systems are thus performed the same way, coupled, in the same tube under the same reaction conditions. During transcription, the 5xe2x80x2 end of the RNA becomes available for ribosomal binding and undergoes translation while its 3xe2x80x2 end is still being transcribed. This early binding of ribosomes to the RNA maintains transcript stability and promotes efficient translation. This bacterial translation system gives efficient expression of either prokaryotic or eukaryotic gene products in a short amount of time. Use of E.coli extract also eliminates cross-reactivity or other problems associated with endogenous proteins in eukaryotic lysates.
Following long-standing patent law, the words xe2x80x9caxe2x80x9d and xe2x80x9can,xe2x80x9d when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d in the claims or specification, denote one or more.
Nucleases
Nucleases are capable of degrading ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA). The nucleases may specifically degrade RNA or DNA, or may be non-specific nucleases, such as S1 nuclease and micrococcal nuclease, and degrade both RNA and DNA. The nucleases encompassed by the present invention include exonucleases and endonucleases.
Ribonucleases (RNases)
Ribonucleases that are inhibited using the present invention include, but are not limited to, RNase A, RNase B, RNase C, RNase 1, RNase T1, micrococcal nuclease, S1 nuclease, or DNase 1. Additional eukaryotic ribonucleases may be inactivated, such as mammalian ribonuclease 1 family, ribonuclease 2 family, mammalian angiogenins, RNase H family, RNase L, eosinophil RNase, messenger RNA ribonucleases (5xe2x80x2-3xe2x80x2 Exoribonucleases, 3xe2x80x2-5xe2x80x2 Exoribonucleases), decapping enzymes and deadenylases. Additional ribonucleases that may be inhibited and/or inactivated by the methods and compositions of the present invention include E. coli endoribonucleases (RNase P, RNase III, RNase E, RNase I,I*, RNase HI, RNase HII, RNase M, RNase R, RNase IV, F; RNase P2,O, PIV, PC, RNase N), E. coli exoribonucleases (RNase II, PNPase, RNase D, RNase BN, RNase T, RNase PH, OligoRNase, RNase R), RNase Sa, RNase F1, RNase U2, RNase Ms, and RNase St. Both endonucleases and exonucleases can be inhibited by the nuclease inhibitor cocktail of the present invention. One of skill in the art can readily employ the methods and compositions of the present invention to inhibit and/or inactivate other RNases known in the art beyond those specifically named herein.
Deoxyribonucleases (DNases)
Deoxyribonucleases that can be inhibited and/or inactivated using the present invention include, but are not limited to, DNase 1, S1 nuclease, and micrococcal nuclease. The nuclease inhibitor cocktail of the present invention can be used to inhibit both endonucleases and exonucleases. One of skill in the art can readily employ the methods and compositions of the present invention to inhibit and/or inactivate other DNases known in the art beyond those specifically named herein.
Compositions
The compositions to which the present methods may be applied in order to inhibit and/or inactivate nucleases will be generally in a liquid form, although a solid composition, such as a matrix comprising immobilized nuclease inhibitor, is also contemplated within the scope of the present invention. If liquid, the composition may be, for example, a reagent used in molecular biology. Representative reagents that may be employed in the present invention include, but are not limited to, water, tris-ethylenediamine tetraacetic acid buffer (TE buffer), sodium chloride/sodium citrate buffer (SSC), 3-(N-morpholinol) propanesulfonic acid (MOPS), Tris buffer, ethylenediamine tetraacetic acid, nucleic acid hybridization buffer, sodium acetate buffer, DNase I digestion buffer, transcription buffer, reverse transcription buffer, cell free extract for in vitro translation, in situ hybridization buffer, and nucleic acid storage buffer/solution. One of skill in the art will understand that the methods of the present invention can be employed with compositions in addition to those named above.
Anti-Nuclease Antibodies
The anti-nuclease antibodies employed in the present invention may be anti-ribonuclease antibodies or anti-deoxyribonuclease antibodies. The anti-ribonuclease antibodies may be antibodies that inhibit one or more of the following ribonucleases: RNase A, RNase B, RNase C, RNase 1, RNase T1, micrococcal nuclease, S1 nuclease, mammalian ribonuclease 1 family, ribonuclease 2 family, mammalian angiogenins, RNase H family, RNase L, eosinophil RNase, messenger RNA ribonucleases (5xe2x80x2-3xe2x80x2 Exoribonucleases, 3xe2x80x2-5xe2x80x2 Exoribonucleases), decapping enzymes, deadenylases, E. coli endoribonucleases (RNase P, RNase III, RNase E, RNase I,I*, RNase HI, RNase HII, RNase M, RNase R, RNase IV, F; RNase P2,O, PIV, PC, RNase N), E. coli exoribonucleases (RNase II, PNPase, RNase D, RNase BN, RNase T, RNase PH, OligoRNase, RNase R), RNase Sa, RNase F1, RNase U2, RNase Ms, and RNase St. Antibodies to additional RNases not specifically disclosed herein can also be employed in the present invention to inhibit and/or inactivate those RNases, or other RNases.
The anti-nuclease antibodies employed in the present invention may also be anti-deoxyribonuclease antibodies that inhibit one or more of the following deoxyribonucleases: DNase 1, S1 nuclease, and micrococcal nuclease. DNases will often require the presence of cations, such as Mg+2 or Ca+2. Antibodies to additional DNases not specifically disclosed herein can also be employed in the present invention to inhibit and/or inactivate those DNases.
The anti-nuclease antibodies may be present in a concentration of up to 100 mg/ml, more preferably in a concentration of up to 50 mg/ml, and even more preferably in a concentration of up to 20 mg/ml. In some embodiments the anti-nuclease antibodies will be present in a concentration of 10 to 0.5 mg/ml. In the most preferred embodiment, the concentration of the anti-nuclease antibodies will be 3 to 4 mg/ml.
For some embodiments of the invention, it will be desired to produce antibodies that bind to a particular nuclease. Means for preparing and characterizing antibodies are well known in the art.
Methods for generating polyclonal antibodies are well known in the art, and a specific method for doing so to generate antibodies to RNase is described in the examples below. Generally, a polyclonal antibody is prepared by immunizing an animal with an antigenic composition and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund""s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund""s adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or in some cases the animal can be used to generate monoclonal antibodies (MAbs). For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from whole cells and blood clots. The serum may be used as is for various applications or the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody or a peptide bound to a solid matrix.
Monoclonal antibodies (MAbs) may be readily prepared through use of well-known techniques. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified expressed protein, polypeptide or peptide. The immunizing composition is administered in a manner that effectively stimulates antibody producing cells.
The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. Mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
The animals are injected with antigen as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund""s complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5xc3x97107 to 2xc3x97108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and have enzyme deficiencies that render them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus, polyethylene glycol (PEG), such as 37% (v/v) PEG, and other compounds are known in the art. The use of electrically induced fusion methods is also appropriate.
Fusion procedures usually produce viable hybrids at low frequencies, about 1xc3x9710xe2x88x926 to 1xc3x9710xe2x88x928. However, this low frequency does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and thus they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
Large amounts of the monoclonal antibodies of the present invention may also be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals that are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
Non-Antibody Nuclease Inhibitors
The invention also envisions the use of non-antibody compounds that function to inhibit nucleases. Suitable such non-antibody nuclease inhibitors include, but are not limited to, diethyl pyrocarbonate, ethanol, formamide, guanidinium thiocyanate, vanadyl-ribonucleoside complexes, macaloid, sodium dodecylsulfate (SDS), ethylenediamine tetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), xcex2-mercaptoethanol, cysteine, dithioerythritol, urea, polyamines (spermidine, spermine), detergents (sodium dodecylsulfate, NP 40, Tween 20, Triton X-100), tris (2-carboxyethyl) phosphene hydrochloride (TCEP), and divalent cations (Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, Cu+2). Using the information provided in this specification, one of skill in the art will be able to identify additional compounds that may be employed in practicing the present invention.
Non-ionic detergents (NP 40, Tween 20, Triton X-100) are not inhibitory of nucleases per se, but have a synergistic effect with anti-nuclease antibodies to enhance the activity of the anti-nuclease antibodies.
Anti-Nuclease Cocktail
As indicated above, the nuclease inhibitor cocktail of the present invention comprises a combination of at least two nuclease inhibitors. Preferably, the nuclease inhibitor cocktail of the present invention remains active over a broad range of conditions. In a preferred embodiment, the nuclease inhibitor cocktail is active in the presence or absence of DTT. In the most preferred embodiment, DTT can be added up to 200 mM without affecting the activity of anti-nuclease antibodies in the cocktail. A preferred embodiment can be used in a broad functional temperature range, including from 4xc2x0 C. to 65xc2x0 C., and at a pH of from 5.5 to 8.5. A preferred embodiment will remain effective at inhibiting nucleases in the presence of guanidinium thiocyanate up to 3M, and of urea up to 6M.